I ligated ORF of size 1.2 Kb in pET-28a-c(+) vector and cloned in DH5 alpha strain of E.Coli. Restriction release of 1.2 Kb was observed. later i transformed it in Expression strain BL-21 and selection was done on chloramphenicol plus kanamycin plate, positive colony was picked and colony PCR confirmed the size of insert. Plasmid Isolated from positively transformed colony had 3 distinct band, on restriction with BamHI and XhoI yielded the insert fragment but we observed three distinct band which is of insert+Vector; Insert and Vector size. I tried several dilution for complete restriction with enzyme with conventional Neb enzyme as well as with Fast digest but complete digestion was not observed.
I went for optimization of induction with different concentration of IPTG. we observed the protein of 47 kDa which was expected but it was also present in control.
Any suggestion in troubleshooting is highly appreciated.
can anyone tell me the number of Band observed in BL-21 strain?
Please Suggest me the reason for incomplete restriction site?
Confirm your clone by sequencing. Use promoter specific primer to get the whole ORF. Make sure your ORF is in frame with the translation site and there is no mutation to create a stop codon. Having insert of right size after restriction digestion is not sufficient to confirm the success of cloning. Why are you using chloromphenicol?? Is it a Rosetta strain? If you are using bl21, use only kanamycin. You do not need to do colony PCR or isolate plasmid once you transformed it into bl21. Simply after you get the colony, inoculate it for expression.
Hi Ashok,
First completely sequence your insert and restriction sites to make sure that you have correct insertion and frame of your gene as Sebastian and Zeyal suggest. Do this from the DH5 cells, once it is confirmed you do not need to check the plasmid again in the BL21 cells , in fact in BL21 it may very well be unstable so transform into BL21(DE3) and use for expression within a week. Adding 1% glucose to the plates will increase plasmid stability by repressing any basal expression.
Secondly make sure that you are using BL21 (DE3) and not just BL21. The DE3 lysogen contains the T7 polymerase under an IPTG inducible prtomoter, without this there will be no T7 polymerase to transcribe from your pET plasmid.
Thirdly, why chloramphenicol? BL21 and BL21(DE3) do not have resistance to chloramphenicol unless you are using Rosetta (DE3) cells where the chlor maintains the pRARE plasmids.
Good Luck
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